Repurposing a tricyclic antidepressant in tumor and metabolism disease treatment through fatty acid uptake inhibition.

Fatty acid uptake is essential for cell physiological function, but detailed mechanisms remain unclear. Here, we generated an acetyl-CoA carboxylases (ACC1/2) double-knockout cell line, which lacked fatty acid biosynthesis and survived on serum fatty acids and was used to screen for fatty acid uptake inhibitors. We identified a Food and Drug Administration-approved tricyclic antidepressant, nortriptyline, that potently blocked fatty acid uptake both in vitro and in vivo. We also characterized underlying mechanisms whereby nortriptyline provoked lysosomes to release protons and induce cell acidification to suppress macropinocytosis, which accounted for fatty acid endocytosis. Furthermore, nortriptyline alone or in combination with ND-646, a selective ACC1/2 inhibitor, significantly repressed tumor growth, lipogenesis, and hepatic steatosis in mice. Therefore, we show that cells actively take up fatty acids through macropinocytosis, and we provide a potential strategy suppressing tumor growth, lipogenesis, and hepatic steatosis through controlling the cellular level of fatty acids.

Cancer cell autophagy, reprogrammed macrophages, and remodeled vasculature in glioblastoma triggers tumor immunity.

Glioblastoma (GBM) is poorly responsive to therapy and invariably lethal. One conceivable strategy to circumvent this intractability is to co-target distinctive mechanistic components of the disease, aiming to concomitantly disrupt multiple capabilities required for tumor progression and therapeutic resistance. We assessed this concept by combining vascular endothelial growth factor (VEGF) pathway inhibitors that remodel the tumor vasculature with the tricyclic antidepressant imipramine, which enhances autophagy in GBM cancer cells and unexpectedly reprograms immunosuppressive tumor-associated macrophages via inhibition of histamine receptor signaling to become immunostimulatory. While neither drug is efficacious as monotherapy, the combination of imipramine with VEGF pathway inhibitors orchestrates the infiltration and activation of CD8 and CD4 T cells, producing significant therapeutic benefit in several GBM mouse models. Inclusion up front of immune-checkpoint blockade with anti-programmed death-ligand 1 (PD-L1) in eventually relapsing tumors markedly extends survival benefit. The results illustrate the potential of mechanism-guided therapeutic co-targeting of disparate biological vulnerabilities in the tumor microenvironment.

Differences in Cerebral Distribution between Imipramine and Paroxetine via Membrane Transporters at the Rat Blood-Brain Barrier.

PURPOSE: The present study aimed to elucidate the transport properties of imipramine and paroxetine, which are the antidepressants, across the blood-brain barrier (BBB) in rats. METHODS: In vivo influx and efflux transport of imipramine and paroxetine across the BBB were tested using integration plot analysis and a combination of brain efflux index and brain slice uptake studies, respectively. Conditionally immortalized rat brain capillary endothelial cells, TR-BBB13 cells, were utilized to characterize imipramine and paroxetine transport at the BBB in vitro. RESULTS: The in vivo influx clearance of [3H]imipramine and [3H]paroxetine in rats was determined to be 0.322 mL/(min·g brain) and 0.313 mL/(min·g brain), respectively. The efflux clearance of [3H]imipramine and [3H]paroxetine was 0.380 mL/(min·g brain) and 0.126 mL/(min·g brain), respectively. These results suggest that the net flux of paroxetine, but not imipramine, at the BBB in vivo was dominated by transport to the brain from the circulating blood. The uptake of imipramine and paroxetine by TR-BBB13 cells exhibited time- and temperature-dependence and one-saturable kinetics with a Km of 37.6 µM and 89.2 µM, respectively. In vitro uptake analyses of extracellular ion dependency and the effect of substrates/inhibitors for organic cation transporters and transport systems revealed minor contributions to known transporters and transport systems and the difference in transport properties in the BBB between imipramine and paroxetine. CONCLUSIONS: Our study showed the comprehensive outcomes of imipramine and paroxetine transport at the BBB, implying that molecular mechanism(s) distinct from previously reported transporters and transport systems are involved in the transport.

Stir bar sorptive-dispersive microextraction by a poly(methacrylic acid-co-ethylene glycol dimethacrylate)-based magnetic sorbent for the determination of tricyclic antidepressants and their main active metabolites in human urine.

A poly(methacrylic acid-co-ethylene glycol dimethacrylate)-based magnetic sorbent was used for the rapid and sensitive determination of tricyclic antidepressants and their main active metabolites in human urine. This material was characterized by magnetism measurements, zeta potential, scanning electron microscopy, nitrogen adsorption-desorption isotherms, and thermogravimetric analysis. The proposed analytical method is based on stir bar sorptive-dispersive microextraction (SBSDME) followed by liquid chromatography-tandem mass spectrometry. The main parameters involved in the extraction step were optimized by using the response surface methodology as a multivariate optimization method, whereas a univariate approach was employed to study the desorption parameters. Under the optimized conditions, the proposed method was properly validated showing good linearity (at least up to 50 ng mL-1) and enrichment factors (13-22), limits of detection and quantification in the low ng L-1 range (1.4-7.0 ng L-1), and good intra- and inter-day repeatability (relative standard deviations below 15%). Matrix effects were observed for the direct analysis of urine samples, but they were negligible when a 1:1 v/v dilution with deionized water was performed. Finally, the method was successfully applied to human urine samples from three volunteers, one of them consuming a prescribed drug for depression that tested positive for clomipramine and its main active metabolite. Quantitative relative recoveries (80-113%) were obtained by external calibration. The present work expands the applicability of the SBSDME to new analytes and new types of magnetic sorbents.

Metabolism and bioactivation of the tricyclic antidepressant amitriptyline in human liver microsomes and human urine.

AIM: Amitriptyline is a widely used tricyclic antidepressant, but the metabolic studies were conducted almost 20 years ago using high-performance liquid chromatography coupled with ultraviolet detector or radiolabeled methods. RESULTS: First, multiple ion monitoring (MIM)- enhanced product ion (EPI) scan was used to obtain the diagnostic ions or neutral losses in human liver microsome incubations with amitriptyline. Subsequently, predicted multiple reaction monitoring (MRM)-EPI scan was used to identify the metabolites in human urine with the diagnostic ions or neutral losses. Finally, product ion filtering and neutral loss filtering were used as the data mining tools to screen metabolites. Consequently, a total of 28 metabolites were identified in human urine after an oral administration using LC-MS/MS. CONCLUSION: An integrated workflow using LC-MS/MS was developed to comprehensively profile the metabolites of amitriptyline in human urine, in which five N-acetyl-l-cysteine conjugates were characterized as tentative biomarkers for idiosyncratic toxicity.

Role of human UGT2B10 in N-glucuronidation of tricyclic antidepressants, amitriptyline, imipramine, clomipramine, and trimipramine.

The role of human UDP glucuronosyltransferase (UGT) 2B10 in the N-glucuronidation of a number of tricyclic antidepressants was investigated and compared with that of UGT1A4 in both the Sf9 expressed system and human liver microsomes. The apparent K(m) (S(50)) values for the formation of quaternary N-glucuronides of amitriptyline, imipramine, clomipramine, and trimipramine were 2.60, 16.8, 14.4, and 11.2 microM in UGT2B10 and 448, 262, 112, and 258 microM in UGT1A4, respectively. The kinetics of amitriptyline and imipramine glucuronidation in human liver microsomes exhibited a biphasic character, where the high- and low-affinity components were in good agreement with our results in expressed UGT2B10 and UGT1A4, respectively. The kinetics of clomipramine and trimipramine glucuronidation in human liver microsomes were sigmoidal in nature, and the S(50) values were similar to those found for expressed UGT1A4. The in vitro clearances (CL(int) or CL(max)) were comparable between UGT2B10 and UGT1A4 for glucuronidation of imipramine, clomipramine, and trimipramine, whereas CL(int) of amitriptyline glucuronidation by UGT2B10 was more than 10-fold higher than that by UGT1A4. Nicotine was found to selectively inhibit UGT2B10 but not UGT1A4 activity. At a low tricyclic antidepressant concentration, nicotine inhibited their glucuronidation by 33 to 50% in human liver microsomes. Our results suggest that human UGT2B10 is a high-affinity enzyme for tricyclic antidepressant glucuronidation and is likely to be a major UGT isoform responsible for the glucuronidation of these drugs at therapeutic concentrations in vivo.

Simultaneous HPLC determination of 14 tricyclic antidepressants and metabolites in human plasma.

A HPLC method has been developed for the simultaneous determination of seven tricyclic antidepressants (TCAs) and seven metabolites in human plasma. The analyte separation was obtained using a C8 reversed phase column and a mobile phase composed of 68% aqueous phosphate buffer at pH 3.0 and 32% ACN. The UV detector was set at 220 nm and loxapine was used as the internal standard. A careful pre-treatment procedure for plasma samples was developed, using SPE on C2 cartridges, which gives satisfactory extraction yields (>80%) and good sample purification. The LOQs were always lower than 9.1 ng/mL and the LODs always lower than 3.1 ng/mL for all analytes. The method was successfully applied to plasma samples from depressed patients undergoing therapy with one or more TCA drugs. Precision data (RSD <8.1%), as well as accuracy results (recovery >80%), were satisfactory and no interference from other drugs was found. Hence the method seems to be suitable for the therapeutic drug monitoring of patients treated with TCAs under monotherapy or polypharmacy regimens.

Efficacy of the MCHR1 antagonist N-[3-(1-{[4-(3,4-difluorophenoxy)phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847) in mouse models of anxiety and depression following acute and chronic administration is independent of hippocampal neurogenesis.

Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a role in the modulation of food intake and mood. In rodents, the actions of MCH are mediated via the MCHR1 receptor. The goal of this study was to investigate the effects of acute (1 h) and chronic (28 days) p.o. dosing of a novel MCHR1 antagonist, N-[3-(1-{[4-(3,4-difluorophenoxy)-phenyl]methyl}(4-piperidyl))-4-methylphenyl]-2-methylpropanamide (SNAP 94847), in three mouse models predictive of antidepressant/anxiolytic-like activity: novelty suppressed feeding (NSF) in 129S6/SvEvTac mice and light/dark paradigm (L/D) and forced swim test (FST) in BALB/cJ mice. A significant increase in the time spent in the light compartment of the L/D box was observed in response to acute and chronic treatment with SNAP 94847. An anxiolytic/antidepressant-like effect was found in the NSF test after acute and chronic treatment, whereas no effect was observed in the FST. Because neurogenesis in the dentate gyrus has been shown to be a requirement for the effects of antidepressants in the NSF test, we investigated whether neurogenesis was required for the effect of SNAP 94847. We showed that chronic treatment with SNAP 94847 stimulated proliferation of progenitors in the dentate gyrus. The efficacy of SNAP 94847 in the NSF test, however, was unaltered in mice in which neurogenesis was suppressed by X-irradiation. These results indicate that SNAP 94847 has a unique anxiolytic-like profile after both acute and chronic administration and that its mechanism of action is distinct from that of selective serotonin reuptake inhibitors and tricyclic antidepressants.

The influence of P-glycoprotein on cerebral and hepatic concentrations of nortriptyline and its metabolites.

The impact of P-glycoprotein (P-gp) on the distribution of nortriptyline (NT) and its metabolites between brain, liver and serum was studied experimentally. The interaction of NT with P-gp in vitro was confirmed by measurement of P-gp stimulated ATPase activity (Km = 257.6 microM, Vmax = 51.0 nmol phosphate released/mg protein-min). Administration of NT (5 mg/kg, s.c.) to mdrla knockout mice resulted in enhanced brain-serum (1.6-fold, p = 0.012) and liver-serum (1.4-fold, p = 0.019) ratios, as compared to the wild-type mice. For a series of NT doses (2.5, 5, 10, 25, 30 mg/kg, i.p.) inhibition of P-gp with cyclosporine A (CsA, 200 mg/kg, i.p.) in rats led to NT brain- and liver-serum ratios that were on average 1.3- (p = 0.005) and 2.1- (p = 0.001) fold higher than those of the controls, respectively. Verapamil (50 mg/kg) (NT, 5 mg/kg) increased the ratios by a factor of 1.6 (p <0.001) and 10.3 (p <0.001) for brain and liver, respectively. Finally, co-administration of methadone (1 mg/kg) did not alter the brain-serum ratio of NT, but in the liver a slight increase (1.5-fold, p = 0.035) was observed. In conclusion, verapamil yielded complete inhibition of P-gp at the blood-brain barrier and CsA had an effect corresponding to about 50% inhibition. The results show that P-gp influences the penetration of NT into the brain, and that drug-drug interactions may take place.

Microsomal binding of amitriptyline: effect on estimation of enzyme kinetic parameters in vitro.

The effect of binding of amitriptyline to human liver microsomes and to microsomes from human B-lymphoblastoid cells on the estimation of enzyme kinetic parameters describing N-demethylation to nortriptyline was investigated using a combination of microsomal binding and in vitro enzyme kinetic studies. Quantitative binding in both matrices increased with higher microsomal protein concentrations (free fractions 0.88-0.32 at 100-500 microg protein/ml in human liver microsomes and 0.82-0.26 at 250-1000 microg protein/ml in microsomes from B-lymphoblastoid cells) and was independent of amitriptyline concentration over a concentration range of 0.2 to 200 microM. Addition of heat-inactivated microsomal protein (50-450 microg/ml) to native human liver microsomes (50 microg/ml) reduced the amitriptyline N-demethylation rate in a protein concentration dependent manner. This effect was greater at lower substrate concentrations and was overcome by saturating concentrations of substrate, thereby decreasing the apparent affinities of the high- and low-affinity components of the N-demethylation process, with minimal effect on the net V(max). Addition of metabolically inactive microsomes from untransfected human lymphoblastoid cells (750 microg/ml) to CYP2C19 (250 microg/ml protein) increased the apparent K(m) value for amitriptyline N-demethylation by 3.5-fold and increased the uncompetitive substrate inhibition constant (K(s)) by 2.2-fold, making substrate inhibition essentially undetectable. A similar effect was seen with CYP3A4, with a 1.8-fold increase in the S(50) (substrate concentration at which half-maximal velocity of a Hill enzyme is achieved). Microsomal binding did not alter the V(max) of either CYP isoform to any appreciable extent. These findings emphasize the importance of incorporating microsomal binding in the estimation of enzyme kinetic parameters in vitro and making appropriate corrections for unbound drug concentrations.

Antidepressivos triciclicos no tratamento farmacologico dos transtornos depressivos em idosos / Guidelines for a rate prescription of tricyclic antidepressants for the treatment of depressive disorders in the elderly

Os antidepressivos triciclicos sao medicacoes eficazes com idosos. Sao drogas relativamente seguras se bem indicadas e manejadas, respeitando-se as limitacoes de seu uso em pacientes geriatricos, bem como suas contra-indicacoes clinicas e farmacologicas. Nessa faixa etaria, a escolha da medicacao antidepressiva torna-se particularmente importante, levando-se em conta que o idoso portador de transtorno depressivo e candidato a uso continuado de antidepressivos...

Five distinct human cytochromes mediate amitriptyline N-demethylation in vitro: dominance of CYP 2C19 and 3A4.

The human cytochromes P450 (CYPs) mediating amitriptyline N-demethylation have been identified using a combination of enzyme kinetic and chemical inhibition studies. Amitriptyline was N-demethylated to nortriptyline by microsomes from cDNA transfected human lymphoblastoid cells expressing human CYPs 1A2, 2C9, 2C19, 2D6, and 3A4. CYP 2E1 showed no detectable activity. While CYP 2C19 and CYP 2D6 showed high affinity, CYP 3A4 showed low affinity; CYP 2C9 and 1A2 showed intermediate affinities. Based on these kinetic parameters and estimated relative abundance of the different CYPs in human liver, CYP 2C19 was identified as the major amitriptyline N-demethylase at low (therapeutically relevant) amitriptyline concentrations, whereas CYP 3A4 may be more important at higher amitriptyline concentrations. Chemical inhibition studies with ketoconazole and omeprazole indicate that CYP 3A4 is the major amitriptyline N-demethylase at 100 mumol/L amitriptyline, while CYP 2C19 is equally important at a substrate concentration of 5 mumol/L. The CYP 1A2 inhibitor alpha-naphthoflavone and the CYP 2C9 inhibitor sulfaphenazole produced much less inhibition of amitriptyline N-demethylation at both substrate concentrations. Quinidine produced no detectable inhibition. The kinetics of amitriptyline N-demethylation by human liver microsomes were consistent with a two enzyme model, with the high affinity component exhibiting Michaelis Menten kinetics and the low affinity component exhibiting Hill enzyme kinetics. No difference was apparent in the kinetics of amitriptyline N-demethylation in two liver samples with low levels of CYP 2C19 activity compared with two other samples with relatively normal 2C19 activity. This may reflect the importance of higher substrate concentration values in estimation of kinetic parameters in vitro.

Routine measurement of fluoxetine and norfluoxetine by high-performance liquid chromatography with ultraviolet detection in patients under concomitant treatment with tricyclic antidepressants.

A robust and rapid high-performance liquid chromatography (HPLC) method is described for therapeutic drug monitoring of fluoxetine and norfluoxetine in the presence of six frequently-used tricyclic antidepressants and their respective metabolites. Liquid-liquid extraction into n-hexane/acetonitrile is used with reextraction into hydrochloric acid for clean-up. The chromatographic separation is carried out on a CN column. The minimum detectable amount is 3 ng injected on column. In addition to qualitative and quantitative validation data for the assay method, results from patient samples are presented. It is concluded that for patients treated with fluoxetine, therapeutic drug monitoring is valuable for optimizing the therapy.

Binding and competitive inhibition of amine uptake at postsynaptic neurones (transport-P) by tricyclic antidepressants.

1. We have provided evidence for a novel amine uptake process for which prazosin is a substrate in postsynaptic neurones, characterized by a paradoxical increase in accumulation of the radioligand when the concentration of the unlabelled drug is increased above 10(-7) M. This increase is due to activation of a proton-dependent, vacuolar type-ATPase-linked uptake process which is blocked by desipramine but is resistant to reserpine. We have now examined the effects of tricyclic antidepressants on this uptake system in a cell line derived from hypothalamic peptidergic neurones, known to be innervated by noradrenergic nerve terminals in vivo. 2. [3H]-imipramine bound to the cells and was displaced by unlabelled imipramine, desipramine, amitriptyline and nortriptyline. The data fitted a single binding site model. This is the first demonstration of antidepressant binding sites in postsynaptic neurones. 3. There was no increase in the binding of [3H]-imipramine at high concentrations of unlabelled imipramine, suggesting that antidepressants inhibit uptake but are not themselves accumulated by peptidergic gonadotrophin releasing hormone neurones. 4. Accumulation of prazosin was competitively inhibited by antidepressants. Tertiary amines were slightly more potent than secondary amines and the presence of a nitrogen atom in the heterocyclic ring enhanced blocking activity. 5. The affinities of the antidepressants for the uptake process are within the range of plasma concentrations that are observed during therapeutic use of these compounds. Since it is likely that this uptake process has a physiological function, its inhibition by antidepressants may provide a new avenue for investigating the mechanism of action of these compounds.

Dosagem plasmática e metabolismo dos antidepressivos tricíclicos / Plasma measurement and metabolism of tricyclic antidepressant drugs

Neste artigo é exposto sucintamente aspectos do metabolismo dos antidepressivos tricíclicos (desmetilaçao e hipdroxilaçao) que devem ser levados em conta na interpretaçao das dosagens plasmáticas e efeitos clínicos destes medicamentos

Metabolic activation of the new tricyclic antidepressant tianeptine by human liver cytochrome P450.

Incubation of [14C]tianeptine (0.5 mM) with human liver microsomes and a NADPH-generating system resulted in the in vitro covalent binding of a tianeptine metabolite to microsomal proteins. This covalent binding required oxygen and NADPH. It was decreased by piperonyl butoxide (4 mM) by 81%, and SKF 525-A (4 mM) by 87%, two relatively non-specific inhibitors of cytochrome P450, and by glutathione (4 mM) by 70%, a nucleophile. Covalent binding was decreased by 54% in the presence of troleandomycin (0.1 mM), a specific inhibitor of the glucocorticoid-inducible cytochrome P450 IIIA3, but remained unchanged in the presence of quinidine (0.1 mM) or dextromethorphan (0.1 mM), two inhibitors of cytochrome P450 IID6. Preincubation with IgG antibodies directed against cytochrome P450 IIIA3 decreased covalent binding by 65% whereas either preimmune IgG or IgG antibodies directed against P450 IA1, an isoenzyme inducible by polycyclic aromatic compounds, exhibited no significant inhibitory effect. We conclude that tianeptine is activated by human liver cytochrome P450 into a reactive metabolite. This activation is mediated in part by glucocorticoid-inducible isoenzymes but not by P450 IID6 (the isoenzyme which oxidizes debrisoquine) nor by P450 IA1 (an isoenzyme inducible by polycyclic aromatic compounds). The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Metabolic activation of the antidepressant tianeptine. II. In vivo covalent binding and toxicological studies at sublethal doses.

Administration of [14C]tianeptine (0.5 mmol/kg i.p.) to non-pretreated hamsters resulted in the in vivo covalent binding of [14C]tianeptine metabolites to liver, lung and kidney proteins; this very high dose (360-fold the human therapeutic dose) depleted hepatic glutathione by 60%, and increased SGPT activity 5-fold. Lower doses (0.25 and 0.125 mmol/kg) depleted hepatic glutathione to a lesser extent and did not increase SGPT activity. Pretreatment of hamsters with piperonyl butoxide decreased in vivo covalent binding to liver proteins, and prevented the increase in SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). In contrast, pretreatment of hamsters with dexamethasone increased in vivo covalent binding to liver proteins, and increased SGPT activity after administration of tianeptine (0.5 mmol/kg i.p.). Nevertheless, liver cell necrosis was histologically absent 24 hr after the administration of tianeptine (0.5 mmol/kg i.p.) to non-pretreated or dexamethasone-pretreated hamsters. In vivo covalent binding to liver proteins also occurred in mice and rats, being increased by 100% in dexamethasone-pretreated animals. In vivo covalent binding to liver proteins was similar in untreated female Dark Agouti rats and in female Sprague-Dawley rats. These results show that tianeptine is transformed in vivo by cytochrome P-450, including glucocorticoid-inducible isoenzymes, into chemically reactive metabolites that covalently bind to tissue proteins. The metabolites, however, exhibit no direct hepatotoxic potential in hamsters below the sublethal dose of 0.5 mmol/kg i.p. The predictive value of this study regarding possible idiosyncratic and immunoallergic reactions in humans remains unknown.

Direct effects of ovarian hormones on antidepressant binding sites.

Tricyclic antidepressants such as imipramine interact with a membrane binding site associated with the uptake of serotonin. Ovarian hormones estradiol and progesterone were found to affect the concentration and affinity of imipramine binding sites in the hypothalamus but not the cortex of female rats. In vivo and in vitro, estradiol increased the amount of imipramine binding at physiological concentrations; at high concentrations estradiol decreased imipramine binding. Ovariectomy (OVX) had no effect on imipramine binding, consonant with the biphasic dose-response relationship for estradiol. The effects of progesterone were dependent upon the concentration of estradiol. Effects of physiological concentrations of both estradiol and progesterone on imipramine binding in an isolated membrane preparation suggest that the hormones are affecting this aspect of serotonergic neuronal function by nongenomic mechanisms.